Project Summary/Abstract Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that infects CD4+ T cells of the immune system. If left untreated, HIV-1 infected individuals will progress to AIDS and may ultimately die as a result. Combination antiretroviral therapy is extremely effective at stopping the replication of HIV-1 in infected individuals. Despite the success of this therapy at suppressing HIV-1 replication to clinically undetectable levels, antiretroviral therapy is not curative. This is due to the persistence of HIV-1 in a silent, or latent, state within a subset of CD4+ T cells known as resting memory CD4+ T cells. In this latent state, these infected cells are not targeted by antiretroviral drugs and cannot be eliminated by the immune system. In HIV-1 infected individuals, latently infected CD4+ T cells are found at extremely low frequencies (~1 per million resting memory CD4+ T cells). However, this population of latently infected cells is very stable, demanding that HIV-1 infected individuals remain on antiretroviral therapy indefinitely. Therefore, this population of latently infected CD4+ T cells is the main barrier to curing HIV-1 infection. Developing strategies to eliminate latently infected cells is a major focus of the NIH, NIAID, and the HIV-1 research field. One primary strategy involves the reactivation of latent infected cells, driving their elimination. We need sensitive, validated molecular assays to monitor this reactivation of latently infected cells in patients receiving such therapeutics. The most direct way to quantify reactivation is to measure the induction of intracellular HIV-1 mRNA, yet current methods suffer from critical shortcomings. Accelevir Diagnostics, LLC is developing a new quantitative assay to measure intracellular HIV-1 mRNA in infected patients. Importantly, this assay will cover globally distributed HIV-1 subtypes. Broadly, this proposal aims to optimize the assay, perform key assay validation studies, and define baseline intracellular HIV-1 mRNA values for ART- suppressed individuals. The goal of this proposal is to develop an optimized commercial prototype, with accompanying standard operating procedures, and set the stage for rapid analytical validation and market entry. !